Concerted versus stepwise mechanism in thymidylate synthase
Zahidul Islam1, Timothy S Strutzenberg, Ilya Gurevic
1Department of Chemistry, The University of Iowa , Iowa City, Iowa 52242-1727, United States.
Journal of the American Chemical Society
|June 21, 2014
Summary
This study investigates the thymidylate synthase (TSase) reaction mechanism. Findings support a concerted mechanism for hydride transfer, highlighting the role of active site arginine in catalysis.
Area of Science:
- Biochemistry
- Enzymology
- Chemical Biology
Background:
- Thymidylate synthase (TSase) is crucial for DNA synthesis and a target for drugs.
- Two mechanisms, stepwise and concerted, are proposed for TSase's rate-limiting hydride transfer step.
- Previous evidence from intermediates and computational studies was contradictory.
Purpose of the Study:
- To experimentally determine the mechanism of the hydride transfer step in TSase catalysis.
- To investigate the role of specific active site residues in the reaction.
Main Methods:
- Utilized secondary kinetic isotope effects (KIE).
- Employed mutagenesis studies.
- Performed primary KIE measurements.
Main Results:
- Experimental data strongly support a concerted mechanism for the hydride transfer.
- Identified a critical role for an active site arginine residue.
- Demonstrated arginine's involvement in substrate binding, nucleophile activation, and hydride transfer.
Conclusions:
- The hydride transfer in TSase catalysis occurs via a concerted mechanism.
- Active site arginine is essential for TSase catalytic efficiency.
- Understanding this mechanism can inform drug design and biomimetic catalyst development.
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